JP3444191B2 - Transcription factors that regulate the phenylpropanoid biosynthetic pathway - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a technique for controlling the expression of genes involved in phenylpropanoid biosynthesis.

[0002]

2. Description of the Related Art With the recent development of plant molecular biology, it is possible to cultivate plant varieties having useful traits such as pest resistance and herbicide resistance by using sense genes and antisense genes. It's coming. That is, a gene involved in the expression of a desired trait is linked to a promoter that can be expressed in a plant in a sense direction or an antisense direction to create a chimeric gene, and by introducing this into a plant as a vector, the desired trait is expressed. Promotes or suppresses the expression of. At present, applying such a method, for example, Bac
Insecticidal B from illus thuringensis
Pest-resistant plants into which the T toxin gene has been introduced in the sense direction (DA Fischhoff et al., Bio / Techn
logic, vol. 232, p. 738-743, 1
987) or a polygalacturonase gene relating to overripening of tomato fruit in the antisense direction and having a good shelf life (CJ Smith et al., Nature, vo).
l. 334, p. 724-727, 988) and the like.

When such a technique is used, in a plant body into which a sense gene (a gene that expresses a target trait and is linked to the promoter in the sense direction) is introduced, the expression of the target trait is usually expressed. And the antisense gene (the same gene as the sense gene,
This suppresses the expression of the target trait in plants into which a gene (a gene linked to the promoter in the antisense direction) was introduced. The expression of the target trait is suppressed by the introduction of the antisense gene because the RNA produced using this gene as a template is complementary to the mRNA derived from the plant original gene involved in the expression of the target trait in plant cells. To inhibit subsequent protein synthesis.

However, many genes of plants form a small-gene family, and the genes forming these families show very high nucleotide sequence homology with each other. Therefore, even if the expression of such a gene is attempted to be suppressed by the antisense method, the RNA of the antisense gene is the mRNA of many other genes forming this small multigene family.
Since they bind to each other at random and suppress their expression, it is not possible to suppress only the expression of the target gene, and various patterns of phenotypic repression are caused, which is completely different from the expected result. Could occur.

On the other hand, the phenylpropanoid biosynthetic pathway is a complex branched reaction system that is specifically present in plants, and is composed of cell wall components such as lignin and suberin.
It is involved in flower pigments and biosynthesis of antibacterial substances. Further, the phenylpropanoid derivative obtained through such a biosynthetic pathway can also be used as a UV protective agent, an insect repellent and the like. Therefore, if the expression of genes involved in this phenylpropanoid biosynthetic pathway can be accurately promoted / suppressed, this biosynthetic pathway can be controlled to produce trees with low lignin content and mass production of useful substances. Also in this case, it was difficult to control the gene by the antisense method due to the above-mentioned problem of homology between genes. For example, a transgenic plant into which the genes for phenylalanine ammonia lyase (PAL) and peroxidase (PRX), which are enzymes that act in the phenylpropanoid biosynthetic pathway, are introduced in the antisense direction has multifaceted inhibitory effects such as growth inhibition. Show (MM Campbell and
R. R. Sederoff, Plant Physio
l. , Vol. 110 ,. p. 3-13, 1996),
In addition, it was reported that the expected change in the lignin content was not detected in tobacco in which the caffeic acid O-methyltransferase gene was introduced in the antisense direction (W. Ni et al., Transge.
n. Res. , Vol. 3, p. 120-126, 19
94).

[0006]

In view of the above problems of the prior art, the present invention aims at appropriately promoting / suppressing the expression of a specific gene involved in the phenylpropanoid biosynthetic pathway in plants. It is intended to provide a DNA and a vector that can be obtained.

[0007]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that a specific group of genes involved in the phenylpropanoid biosynthetic pathway, namely cinnamyl alcohol dehydrogenase (CAD). ) Gene, chalcone acid synthetase (CHS) gene, 4-coumaric acid CoA ligase (4CL) gene, PAL gene, PR
Focusing on the fact that a sequence controlling its expression exists in the 5'upstream untranslated region of the X gene and the like, and that the sequence has extremely high homology between these different genes, A factor that binds to a common sequence and promotes transcription of these genes (hereinafter simply referred to as a transcription factor)
It was found that the expression of these genes can be surely promoted and suppressed by isolating the gene of the present invention and introducing the DNA encoding the same into a plant as a sense gene or an antisense gene, and completed the present invention.

That is, the above-mentioned object of the present invention is to provide a DNA encoding this transcription factor, specifically a DNA comprising the nucleotide sequence shown in SEQ ID NO: 1, which hybridizes with this DNA under stringent conditions and which is phenylpropanoic. It is solved by DNAs encoding transcription factors that control the biodegradation pathway of the noids and recombinant vectors containing these DNAs. In addition, the stringent conditions here include 6 × SSC (0.9 M NaCl, 0.09
M sodium citrate), temperature 55 ° C.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is described in detail below.

Since the phenylpropanoid biosynthetic pathway is widely conserved in common in the plant kingdom, there are many genes involved in this, such as the CAD gene, and transcription factors that control the expression of these genes. Plants have in common. Therefore, the DNA of the present invention can be isolated from many plants according to a standard method regardless of whether it is herbaceous or woody. Further, the DNA of the present invention is obtained by the phosphite triester method (H. Hunkapiller et al., Nature.
e, vol. 310, p. 105-111, 1984).
It can also be obtained by chemical synthesis by a general method such as.

The obtained DNA is a plant-expressible promoter such as the cauliflower mosaic virus 35S promoter (CaMV35S promoter),
It is used by being linked in the sense direction or antisense direction to the downstream region of the promoter of nopaline synthetase, the promoter of ribulose diphosphate carboxylase / small subunit of oxygenase and the like. In order to promote the expression of the target gene (in the present invention, a gene to be targeted for promotion of expression or suppression of expression), the DNA of the present invention is linked to a promoter in the sense direction, and conversely,
In order to suppress the expression of the target gene, the DNA of the present invention is ligated to the promoter in the antisense direction. In order to promote the expression of the target gene, the DNA of the present invention linked to the promoter in the sense direction and the target gene linked to the downstream region of another promoter are integrated into one vector, or It may be introduced into plant cells as a separate vector.

The DNA ligated to the promoter can be prepared by the microinjection method, electroporation method,
It can be directly introduced into plant cells directly by using polyethylene glycol method, fusion method, high-speed ballistic venelation method, or the like, but it can be introduced into a plant gene transfer plasmid and used as a vector to have plant infectivity. It can also be indirectly introduced into plant cells via viruses or bacteria. Examples of such viruses include:
Cauliflower mosaic virus, gemini virus, tobacco mosaic virus, brom mosaic virus, etc. can be used, and as bacteria, Agrobacterium tumefaciens ( Agrobacterium tum)
efaciens or less, A. Abbreviated as Tumefacience. ), Agrobacterium rhizogenes ( Agro
Bacterium rhizogenes ) etc. can be used. A. When a gene is introduced into a plant by the Agrobacterium method using Tumefaciens, such a plasmid is, for example, pBI10.
1, pBI121 (both from Clontech) or the like can be used.

In the present invention, a plant body in which the expression of a target gene is promoted / suppressed can be obtained by proliferating / redifferentiating a plant cell into which the DNA of the present invention has been introduced by the above method. . Propagation of such plant cells
The conditions for regeneration may be appropriately selected depending on the type of plant.

The DNA of the present invention and a vector incorporating the same are used in the CAD gene, CHS gene, 4CL gene, P
It is possible to promote / suppress expression of AL gene, PRX gene and the like. Furthermore, not only these genes but also any gene in which the common sequence they have is present in the transcriptional regulatory region can be regulated by the DNA and vector of the present invention.

The present invention also promotes gene expression.
The plant that can be suppressed is also not particularly limited. Basically, it is considered that the effect of the present invention can be obtained in any plant having a phenylpropanoid biosynthetic pathway. For example,
The present invention can be applied to not only tobacco but also grass plants such as rice, Arabidopsis, petunia, and wood plants such as poplar, eucalyptus, acacia, cedar, and pine.

[0016]

[Function] Regarding genes involved in the phenylpropanoid biosynthetic pathway, consensus sequences as shown in Table 1 have been found in various plant species (note that the consensus sequences are listed in the column of consensus sequence in the table). Numbers indicate the distance from the transcription start point (unit is bp).

[0017]

[Table 1]

Therefore, the transcription factors that bind to this consensus sequence have almost the same structure in these gene groups, and in addition, the DNAs encoding these transcription factors also have highly homologous base sequences. It is believed to have. Therefore, the DNA of the present invention is specific for all of the CAD gene, CHS gene, 4CL gene, PAL gene, PRX gene and the like having this common sequence, regardless of the type of gene on which the plant or its transcription factor acts, Its expression can be promoted or suppressed.

That is, when the DNA of the present invention is introduced into a plant as a sense gene, in addition to the original transcription factor of the plant, this gene also synthesizes a transcription factor capable of binding to the above-mentioned common sequence in the plant. . This is independent of the type of plant into which this gene has been introduced. As a result, the expression level of the transcription factor is increased as a whole in the plant body, and since these transcription factors bind to the common sequence at a higher frequency, the expression of the gene having this sequence in the transcriptional regulatory region is promoted. That will be the case.

On the other hand, when the DNA of the present invention is introduced into a plant as an antisense gene, in the plant,
The RNA produced using this gene as a template complementarily binds to the mRNA derived from the gene for the transcription factor originally possessed by the plant, and inhibits the synthesis of the transcription factor. As a result, the expression level of the transcription factor decreases, and its binding to the above-mentioned common sequence does not occur easily, so that the expression of the gene having this sequence in the transcriptional regulatory region is suppressed.

[0021]

EXAMPLES The present invention will be described below based on examples.

[Example 1] 1. Isolation and Purification of Tobacco mRNA After seed germination, about 10 g of a tobacco leaf that had been grown in a greenhouse for about 1 month was crushed in the presence of liquid nitrogen, and its total RNA was extracted by the method of Chomczznski et al.

The total RNA obtained was diethyl carbonate 0.2.
After dissolving in sterile water containing 2% and incubating for 5 minutes at a temperature of 65 ° C., a loading buffer (20 mM) of twice the equivalent amount
Tris-Hcl, pH 7.6, 0.1 M NaCl,
1 mM EDTA, 0.1% SDS) was added to dilute,
It was applied to a pre-activated oligo dT cellulose column. The column is then replaced by 5 column volumes.
-10 volumes of loading buffer, and 5 volumes of wash buffer (20 mM Tris-Hcl, pH 7.6,
0.5 M NaCl, 1 mM EDTA, 0.1% S
It was washed with DS). Elution was carried out with an elution buffer (10 mM Tris-Hcl, 2 to 3 times the column volume).
pH 7.6, 1 mM EDTA, 0.05% SDS)
Purified mRN
A10 mg was obtained.

2. Preparation of cDNA Library From the mRNA obtained in 1, the Gubler & Hof
fman's method (Gubler et al., vo
l. 25, p263-269, 1983), and cDNA was synthesized using an oligo dT primer. This c
After inserting the DNA into the Eco RI site of the phage DNA λgt11, the lambda phage coat protein (Gigap
ack II packing extract
s, commercially available from Stratagene. ) With in
Vitro packaging was performed to obtain recombinant lambda phage.

On the other hand, Escherichia coli Y-1090 strain was inoculated into 10 ml of LBMM medium (1% tryptone, 1% sodium chloride, 0.5% yeast extract, 10 mM magnesium sulfate and 0.4% maltose) and incubated at 37 ° C. for 12 hours. It pre-cultured by shaking. After pre-culturing, the cells were collected by centrifugation, and the cells were preliminarily cooled to 4 ° C with 10 mM magnesium sulfate 10
It was suspended in ml to make it susceptible to lambda phage infection.

Infection was carried out by mixing the recombinant E. coli phage thus obtained with the above recombinant lambda phage and leaving it at 37 ° C. for 15 minutes.

3. Transcription factor screening E. coli Y-1090 infected with recombinant lambda phage
Were cultured at 37 ° C. on LBMM medium containing 1.5% agar. Then, after about 4 hours, when plaque generation was observed, a nylon membrane filter (Hybond) which had been soaked in 10 mM isopropyl thiogalactoside (IPTG) and air-dried on the culture plate in advance was observed.
-N (Amersham Co.) was placed and left at 37 ° C. overnight and then removed. The nylon membrane filter was then passed through a binding buffer (10 mM Hepes, pH 7.5, 50 mM N) containing 5% skim milk.
aCl, 1 mM EDTA) at 4 ° C. for 1 hour for blocking, and further soaked in the same binding buffer plus probe for screening,
A binding reaction between this probe and a transcription factor was performed at 4 ° C. for 2 hours.

As a probe for screening, a common sequence (P-BOX sequence: -CCACTTGA) existing in the 5'upstream untranslated region of the kidney 4CL gene or PAL gene, horseradish PRX gene or the like is used as a probe.
GTAC-) was used. That is, a double-stranded oligonucleotide having this P-BOX sequence was synthetically prepared and used by labeling with digoxigenin (DIG).

After the binding reaction, the nylon membrane filter was washed by immersing it in binding buffer three times for 30 minutes at room temperature, and then subjected to primary screening and secondary screening by chemiluminescence detection. As a result, one positive plaque producing a protein that binds to the consensus sequence was detected from 1.0 × 10 ⁶ plaques. In this experiment, the operations from the DIG label of the double-stranded synthetic oligonucleotide to the screening by chemiluminescence detection mainly follow the non-radioisotope DIG-nucleic acid detection method using a commercially available kit (Boehringer Mannheim). went.

4. D encoding a consensus sequence binding protein
Determination of the nucleotide sequence of NA From the phage forming the above positive plaque, D
NA was extracted. The insert portion of the obtained phage DNA (the portion obtained by inserting the cDNA obtained from tobacco in 2) was amplified by PCR using a primer containing the cloning site of λgt11, and agarose gel electrophoresis was performed. The presence of a DNA fragment of about 1 kbp was confirmed. Therefore, after phosphorylating the end of this 1 kbp DNA fragment, it was integrated into the plasmid pNoTA / T7 (PRIME PCR CLONER ^™ CLO).
NING SYSTEM (5prime, 3prim
e, Inc. )use. ), This recombinant plasmid DN
Regarding A, the base sequence of the DNA encoding the title protein was determined by determining the base sequence by the dideoxy method using a DNA sequencer (DNA Sequencer Model 373S (manufactured by Perkin-Elmer)).

The sequence is shown in SEQ ID NO: 1, and it is considered that this protein is composed of about 200 amino acids. The mass is about 25 kDa. In addition, from the homology search of this protein (as a database,
SWISS-PROT Rel. Use 34. ), This protein is presumed to be composed of 2 units of LIM domain, which is a kind of zinc finger motif. If so, this transcription factor
This is the first example showing that the LIM domain binds to DNA. Therefore, this protein was named Ntlim1.

5. Ntli for confirming DNA binding activity
Acquisition of m1 Protein screened as described above, Ntl
In order to confirm the DNA binding ability of im1, it was attempted to produce the required amount of this in E. coli.

First, a DNA fragment (Ntlim1 gene) having the nucleotide sequence shown in SEQ ID NO: 1 is linked to the Ntlim1 translation start codon region or translation stop codon region, and a PC containing a restriction enzyme Bam HI site is used for PC.
It was amplified by the R method. This DNA fragment is used as the restriction enzyme Ba
After digestion with m HI, the expression plasmid pGEX-2T
Bam of K (Pharmacia Biotech)
The orientation of the insert DNA and the correctness of the frame were confirmed by inserting into the HI site and further examining the base sequence of the junction site. When Ntlim1 is produced using such a vector, Ntlim1 will be obtained as a GST fusion protein under the control of the tac promoter.

Then, the prepared expression plasmid was introduced into a competent cell of Escherichia coli JM109 (manufactured by Toyobo Co., Ltd.), and this E. coli was used as an antibiotic for ampicillin 100.
A transformant in which the Ntlim1 gene had been introduced was selected by culturing in LB agar medium containing 1 mg / l (tryptone 1%, salt 0.5% and yeast extract 0.5%). This transformant was inoculated in 1 ml of LB medium and
After overnight preculture at 200 rpm, ampicillin-containing L
B medium (ampicillin concentration 100 mg / l) 30 ml
At 37 ° C. and 200 rpm, shaking culture was performed. Then, when the OD600 reached about 1.0, in the medium,
Add IPTG to a final concentration of 2 mM, then add 3 more
Shaking culture was continued at 7 ° C. and 100 rpm. After 5 hours, the bacterial cells were collected by centrifugation and the PBS buffer solution (14
0 mM NaCl, 2.7 mM Kcl, 10.1 mM
Na ₂ HPO ₄ , 1.8 mM KH ₂ PO ₄ , pH
The cells were suspended in 7.3) and centrifuged again to collect bacterial cells.

The obtained bacterial cells were suspended in 2 ml of PBS buffer solution, and the suspension was subjected to ultrasonic treatment to crush it, followed by centrifugation (15000 g , 15 minutes) to obtain a supernatant (soluble fraction). Min) was separated. S for this soluble fraction
After performing DS-polyacrylamide electrophoresis and confirming that the target protein, that is, the Ntlim1-GST fusion protein was inducibly expressed, the following operation was performed on this fraction.

First, glutathione sepharose 4B (P
10 μl of 50% slurry (Harmcia Biotech) was added to this fraction, stirred, and allowed to stand for 30 minutes at room temperature to adsorb the Ntlim1-GST fusion protein to this glutathione sepharose 4B. Glutathione Sepharose 4B thus adsorbing the target protein was recovered by centrifugation (500 g , 5 minutes),
After washing 3 times with BS buffer, only Ntlim1 was eluted from here and isolated. That is, the collected and washed glutathione Sepharose 4B was mixed with 19 μl of PBS buffer and thrombin protease (1 cleavage unit:
Enzyme amount capable of decomposing 90% of GST fusion protein 100 μg in PBS in 16 hours. ) 1 μl was added and suspended, and the suspension was left at room temperature for 2 hours and then centrifuged (500 g ,
The supernatant was separated by 5 minutes). Then, Ntlim1 was isolated by performing SDS-polyacrylamide electrophoresis on this supernatant.

6. Confirmation of DNA binding activity of Ntlim1 The DNA binding activity was confirmed by gel mobility shift method using Ntlim1 isolated in 5.

2 μg of purified Ntlim1 was added to a binding buffer (10 mM Hepes, pH 7.5, 50 mM Na).
Cl, 1 mM EDTA) 10 μl, and
D containing the P-BOX region used in 3 as a probe
IG-labeled double-stranded synthetic oligonucleotide 10 nmol,
Add 2 μg of salmon sperm DNA as carrier DNA,
It was left at room temperature for 20 minutes.

About this binding reaction solution, 1 × TAE
(6.7 mM Tris-Hcl, pH 7.9, 1 mM
5% with EDTA, 3.3 mM sodium acetate)
Electrophoresis was performed on a polyacrylamide gel at 100 V, which was terminated before free probe flowed out of the gel. Then, the electrophoretic pattern was transferred from the gel to a nylon membrane filter, and chemiluminescence detection was performed in the same manner as in 3. As a result, a band shifted to a position different from the electrophoretic band originally exhibited by Ntlim1 was observed.
The presence of the A-protein complex was confirmed. In addition, this shift band is a non-labeled probe (DI
Except that it is not labeled with G, the probe is exactly the same as that used for detecting the shift band. ) When 1 μmol was further added and electrophoresis was performed under the same conditions, it disappeared. Therefore, it can be seen that the binding between the Ntlim1 protein and the P-BOX sequence is specific.

7. Detection of expression regulation activity by Ntlim1
討 First, to express the Ntlim1 gene in plants, the position of the β- glucuronidase gene of the plasmid pBI221 (Clontech Co.) incorporating a DNA fragment containing the Ntlim1 gene in the sense direction, using this plasmid as an effector. In this effector, a schematic diagram of a portion in which the Ntlim1 gene is integrated is shown as FIG. Also, apart from this, P-BOX
Double-stranded oligo DNA in which the sequence was repeated three times, and P-BOX
Similar to the sequence, the so-called G-BOX sequence (-CCACGTG) commonly present in the 5'untranslated region of the CAD gene and the like.
G-) was repeated 3 times to obtain a double-stranded oligo DNA,
The fusion gene was ligated in tandem so that the OX sequence would be upstream, and further ligated to the upstream side of the β-glucuronidase (GUS) gene that had been inserted into the Eco RV site (-90 bp) of the CaMV35S promoter in advance. Created. Then, this fusion gene was integrated into pUC19, and this plasmid was used as a reporter. That is, in this example, in order to clarify the function of Ntlim1 more simply, in the downstream region of the P-BOX sequence on which it acts, not the CAD gene or the like which should originally exist,
The GUS gene whose expression was extremely easy to detect was placed as a model. In this case, the G-BOX sequence is considered to act as an enhancer. In this reporter, a schematic diagram of the portion in which the fusion gene is integrated is shown in FIG.
Show as.

The two kinds of recombinant plasmids prepared as described above were introduced into competent cells of Escherichia coli JM109 and amplified by culturing the E. coli, and finally about 1 mg of each was obtained. Plasmid DNA was obtained. The culture of E. coli at this time was carried out using an LB medium at a temperature of 37 ° C. and a shaking speed of 200 rpm. The isolation and purification of plasmid DNA from E. coli was performed according to a standard method.

On the other hand, the method of Okada et al. (K. Okada e
t al. , Plant Cell Physiol. ,
Vol. 27, 619, 1986), protoplasts were prepared from BY-2 cultured tobacco cells. Then electroporation buffer (5 mM MES,
30 mM Kcl, 0.3 M mannitol, pH 5.
8) About 3 × 10 ^{6 of} this protoplast suspended in 1 ml
An electric pulse (200 V, 250 μF) was applied to each individual, and 10 μg of the reporter or 10 μg of each of the reporter and the effector was introduced by the electroporation method (using a gene transfer device GenePulser (manufactured by Bio-Rad)). Protoplasts thus treated with gene transfer were washed with 0.4 M mannitol, and cultured in a protoplast medium (mixed salts for Murashige-Skoog medium (manufactured by Nippon Shinyaku Co., Ltd., with 0.4 M mannitol) added) at a temperature of 25 ° C. for 24 hours. And then crushed and subjected to the method of Jefferson et al. (R. Jefferson et al., EMB.
OJ. , Vol. 6, p. 3901-3907, 19
GUS activity was measured according to 97). As a result, in the protoplasts in which the reporter and the effector were simultaneously introduced, the expression of the GUS gene which was about 3 times higher than that in the case where the reporter alone was introduced was detected. This is a protein derived from the effector Ntlim1 gene, Ntl.
The expression of the GUS gene linked to the downstream region of the P-BOX sequence in the reporter was greatly promoted by the effect of im1, that is, this Ntlim1 binds to the P-BOX sequence and the expression of the gene existing in the downstream region is increased. Ntli is a transcription factor that promotes expression and also encodes it.
It is shown that the expression of the gene controlled by Ntlim1 is promoted by introducing the m1 gene as a sense gene into a plant.

[Example 2] 1. Tobacco Sense and Antisense Ntlim1
Using the plasmid pBI121 (Clontech) as a vector for introducing the Ntlim1 gene into a plant, the Ntlim1 gene was added to this plasmid at the same position as in the case of the effector created in 7 of Example 1.
Incorporated in sense or antisense orientation. The orientation of the inserted Ntlim1 gene in the obtained recombinant plasmid was determined by a restriction enzyme digestion experiment, and this was then electroporated into A. Tumefacience E
Introduced into HA105 (apply 2500 V, 25 μF as an electric pulse in 10% glycerol). Then, this A. Tumefaciens 100m kanamycin
Selection was carried out by culturing in LB medium containing g / l at 28 ° C. for 2 days to obtain only individuals into which the recombinant plasmid had been introduced.

On the other hand, examples of plants into which the Ntlim1 gene is introduced include tobacco ( Nicotiana tabacu).
m L cv. SR-1) was used as the material. That is,
Seedlings grown under aseptic conditions (approx. 4 weeks after germination)
The leaves were cut into 5 mm squares, and the resulting leaf pieces were used for the recombinant plasmid introduction A. By dipping the leaf epidermis downward in a culture solution of Tumefaciens for 1 to 3 minutes, the A. The Ntlim1 gene was introduced by infecting Tumefaciens. The leaf pieces after the infection treatment were treated with a sterilized paper towel or the like to remove the adhered culture solution, and then the callus induction medium (Murashige-Skoog basal medium, 3% sucrose, 0.
25% gellan gum, 1 mg / l naphthalene acetic acid, 0.1
(mg / l benzyladenine) and cultured for 3 days under continuous lighting at 25 ° C., then shoot forming medium (Murashige-Skoog basal medium, 3% sucrose, 0.25% gellan gum, 0.1 mg / l naphthalene acetic acid) 1 mg / l benzyladenine, 100 mg / l kanamycin, 500 m
(g / l carbenicillin) and continued to be cultured under the same temperature and light conditions to differentiate the foliage.

The differentiated foliage was cut out after about 4 weeks of culture in a shoot forming medium, and 100 mg of kanamycin
/ L and carbenicillin 500mg / l
The roots were rooted by transplanting to Skoog basal medium (adding sucrose 3%, agar 0.8% or gellan gum 0.25%) and further culturing for about 4 weeks under the same temperature and light conditions. The rooted individual is a Metromix 350 (Scot
ts-Sierrea Horticulture Co
(company) was used as a culture soil and grown in a greenhouse at 25 ° C.

2. Taba introduced with Ntlim1 gene
Analysis of Ko 2-1. Genomic DNA was extracted from the transformed tobacco leaves grown in Analysis 1 by PCR method by a standard method, and PCR was performed using an oligonucleotide corresponding to the nucleotide sequence in the kanamycin resistance enzyme gene as a probe. As a result, amplification of the Ntlim1 gene was detected in all PCR samples, and it was confirmed that all 25 transformants used in the analysis had the sense or antisense Ntlim1 gene (sense transformant 13 Individuals: 12 antisense transformants.).

2-2. Analysis by Northern hybridization method From the transformed tobacco (8-week-old) in which the presence of the Ntlim1 gene was confirmed in 2-1, a sense transformant (in which the Ntlim1 gene was introduced in the sense direction) and antisense transformation. Body (Ntli in antisense direction
Introduced m1 gene. ) Two individuals were selected and total RNA was extracted from their stems by the acid guanidine phenol chloroform method. At the same time, as a control, total RNA was also obtained from untransformed tobacco.
Was extracted.

10 μg of the extracted total RNA was 1.2% containing a final concentration of 0.66 M formaldehyde.
Agarose gel and 1x MOPS buffer (20 mM MOPS
PS / pH 7.0, 5 mM sodium acetate, 0.5 mM
(EDTA) and electrophoresed at 60 V for 2 hours for fractionation. After the migration, the migration pattern was transferred from the gel to a nylon membrane filter, and this nylon membrane filter was subjected to Northern hybridization.

Northern hybridization is performed using Nt.
PCR from genomic DNA of tobacco other than lim1 gene
PAL gene (820 bp) obtained by amplification by the method
And 4CL gene (610 bp) as a probe,
Similar to 3 of Example 1, non-radioisotope DIG-
The nucleic acid detection method was used. FIG. 3 shows the results of chemiluminescence of the above nylon membrane filter after Northern hybridization.

From FIG. 3, Ntlim in the transformant is shown.
Expression of the 1 gene was higher than that of the non-transformant C (lane5) in the sense transformants OP1 and OP2 (lane5).
In 1 and 2), it was strong, and conversely, antisense transformant UP
1 and UP2 (lane3, 4) are weak.
Further, in the sense transformants OP1 and OP2, stronger expression of the PAL gene and the 4CL gene was observed than in the non-transformant C, and this tendency was particularly remarkable in OP2. On the other hand, in the antisense transformant UP2, P
The expression of AL gene and 4CL gene was almost completely suppressed. In the antisense transformant UP1, the expression of the PAL gene and the 4CL gene was not significantly suppressed because the introduced amount of the antisense gene (the expression of the PAL gene and the 4CL gene was almost completely suppressed in this individual). In the antisense transformant UP2,
It is considered that multiple copies of the Ntlim1 antisense gene were introduced into the plant genome. ), And the effect of the position in the plant genome into which it was introduced is considered to have an influence.

From these results, the Ntlim1 gene,
That is, it was revealed that the DNA according to the present invention can promote and suppress the expression of PAL gene and 4CL gene.
The present invention also provides that the transcription factor synthesized from the DNA of the present invention acts by binding to P-BOX of these genes or a similar 5'untranslated region common sequence thereof. Since this is the basic principle, the gene expression / promotion effect by this is not limited to the above-mentioned PAL gene and 4CL gene, and any gene having such a common sequence can be exerted regardless of its type.

[0052]

INDUSTRIAL APPLICABILITY According to the present invention, the expression of a gene having a predetermined common sequence in its 5'untranslated region is specifically promoted.
It became possible to suppress.

That is, according to the present invention, the CAD gene, 4CL gene, PAL gene, P
It is possible to control genes involved in the plant phenylpropanoid biosynthetic pathway such as the RX gene.

Further, since the above CAD and the like are enzymes deeply involved in the lignin biosynthetic pathway among the phenylpropanoid biosynthetic pathways, according to the present invention, which can control the expression of these genes, It enables the development of promising trees as raw materials for paper and pulp, including the production of low-lignin trees.

[Sequence list]

SEQ ID NO: 1 Sequence length: 988 Sequence type: Nucleic acid Sequence type: cDNA to mRNA Origin organism name: Nicotiana ( Nicotiana)
Tobacum ) Tissue type: Leaf array features Symbols representing features: CDS Location: 100. ． 702 Method for determining feature: E Feature symbol: domain Location: 127. ． 282 Method of determining feature: S Other information: LIM domain which is a type of zing finger motif, symbol representing feature: domain Location: 427. ． 582 Method of determining features: S Other information: LIM domain sequence which is a kind of zing finger motif

[Brief description of drawings]

FIG. 1 shows the effector used in Example 1 with N
It is a schematic diagram which shows the part in which the tlim1 gene was integrated.

2] In the reporter used in Example 1, P-
It is a schematic diagram which shows the part in which the fusion gene which has a BOX sequence was integrated.

FIG. 3 is a diagram showing the expression status of various genes in tobacco into which the Ntlim1 gene has been introduced in the sense or antisense direction.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI C12R 1:91) C12N 5/00 C (C12N 15/09 ZNA C12R 1:91) (C12P 21/02 C12R 1:91) ( 56) References JP-A-10-94392 (JP, A) Baltz R. et al. et al. , Pl ant Journal, Vol. 2 (5), pp. 713-721 (1992) (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 15/09 ZNA A01H 5/00 C12N 5/10 C12P 21/02 BIOSIS (DIALOG) WPIDS (STN) SwissProt / PIR / GeneS eq GenBank / EMBL / DDBJ / Gene Seq

Claims (8)

(57) [Claims] 1. A DN comprising the nucleotide sequence shown in SEQ ID NO: 1.
A. 2. A DN comprising the nucleotide sequence shown in SEQ ID NO: 1.
A DNA that hybridizes with A under stringent conditions and that encodes a transcription factor that controls the phenylpropanoid biosynthetic pathway. 3. A recombinant vector having the DNA according to claim 1 or 2. 4. A recombinant vector having the DNA according to claim 1 or 2 in the sense direction. 5. A recombinant vector having the DNA according to claim 1 or 2 in the antisense direction. 6. A plant cell into which the DNA according to claim 1 or 2 is introduced. 7. A plant regenerated from the plant cell according to claim 6. 8. A protein encoded by the DNA according to claim 1 or 2.